Cellular DNA normally exists in a dynamic environment. Cellular functions of repair, recombination, replication, and cell cycle regulation are intimately interwoven to maintain genomic stability and generate genetic diversity (reviewed by Petes et al., 1991, Recombination in yeast, In: The Molecular and Cellular Biology the Yeast Saccharomyces (eds. J. R. Broach, J. R. Pringle, and E. W. Jones), pp. 407-521, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Drapkin et al., 1994, Cell 77:9-12; Kuhn et al., 1995, Genes Dev. 9:193-203; Friedberg, et al. 1995, DNA repair and mutagenesis, pp. 147-192, ASM Press Washington, D. C.; Li et al., 1995, Cell 83:1079-1089). A mutation in a gene whose product is critical to any of these processes may result in a variety of clinical signs that include neurological disorders, immunodeficiency, and predisposition to cancer.
Understanding the molecular mechanisms of repair and recombination will be beneficial to understanding the etiology of diseases associated with defects in these processes. Transgenic animals are useful for studying the dynamic nature of DNA in vivo. In particular, transgenic mice are ideal. The similarities between the genetic constitution and organization of humans and mice are remarkable (Lyon and Searle, 1989, Genetic variants and strains of the laboratory mouse, 2nd ed. Oxford University Press, Oxford). In addition, anatomical similarities between mice and humans provide the opportunity for direct physiological comparison. Targeted disruption of genes encoding protein products such as the p53 tumor suppressor (Donehower et al., 1992, Nature 356:215-221), the mismatch repair proteins (Baker et al., 1995, Cell 82:309-319; de Wind et al., 1995, Cell 82:321-330) and the xeroderma pigmentosa complementation groups (Sands et al., 1995, Nature 377:162-165; de Vries et al., 1995, Nature 377:169-173; Nakane et al., 1995, Nature 377:165-168) have revealed striking similarities to inherited disorders in humans.
A number of different DNA repair pathways are responsible for correcting a variety of specific DNA lesions. These pathways include nucleotide excision repair, mismatch repair and double-strand break (DSB) repair. The mechanisms responsible for nucleotide excision repair and mismatch repair are fairly well understood, and mutations affecting these processes have been characterized (reviewed in Friedberg, 1992 Cell 71:887-889; Cleaver, 1994, Cell 76:1-4). The mechanisms responsible for the repair of DSBs remain poorly understood. Several inherited disorders of mammals feature defects in the repair of DSBs that are associated with hypersensitivity to ionizing radiation and immunodeficiency. These include Ataxia-Telangiectasia (AT) in humans, and autosomal recessive scid (severe combined immunodeficiency) in mice and in horses.
AT is an autosomal recessive defect that results in progressive neurodegeneration, immune deficiencies, susceptibility to cancer, premature aging and permanently dilated blood vessels in the eyes, ears, and parts of the face (reviewed by Lehmann and Carr, 1995, Trends in Genet. 11:375-377). Cells from AT patients may often display chromosomal instability and tend to accumulate chromosomal aberrations. Additionally, "normal" cells temporarily suspend DNA replication after sustaining DNA damage whereas AT cells continue to replicate their DNA in the presence of DNA damage. Cells isolated form AT patients also exhibit an increased rate of intrachromosomal recombination (Meyn, 1993, Science 260:1327-1330) and fail to halt the cell cycle in response to ionizing radiation (Kastan et al., 1992, Cancer Res. 51:6304-6311). AT patients are predisposed to cancer and may be hypersensitive to ionizing radiation (Taylor et al., 1975, Nature 258:427-429). The AT gene was recently cloned (Savitsky et al., 1995, Science 268:1749-1753) and has homology to the phosphatidylinositol (PI) 3-kinases including (reviewed by Zakian, 1995, Cell 82:685-687): Saccharomyces cerevisiae TOR1 and TOR2 (G1-S phase transition), mammalian FRAP and rRAFT (G1-S transition), Schizosaccharomyces pombe RAD3 (rad 3 cells are sensitive to X-ray and ultraviolet light and fail to arrest in G2 after DNA damage), S. cerevisiae MEC1 (required for S-M and G2-M checkpoints and meiotic recombination) and mammalian DNA-PK.sub.cs (the catalytic subunit of DNA-PK which is involved in repairing DSBs generated during V(D)J recombination and after exposure to ionizing radiation; see below). The observed homology to these proteins indicates that ATM plays an important role in cell growth and cell cycle regulation. Although these proteins have homology to lipid kinases, they may function as protein kinases (reviewed by Hunter, 1995, Cell 83:1-4). For example, DNA-PK.sub.cs, has protein kinase activity in vitro, but no lipid kinase activity has been detected (Hartley et al., 1995, Cell 82:849-856).
The scid mutation in mice is recessive and results in an immunodeficiency caused by a failure to repair a specific class of broken DNA ends that arise during rearrangement of T cell receptor (TCR) and immunoglobulin (Ig) genes (V(D)J recombination) in developing lymphocytes (see below). A similar defect has recently been described in Arabian foals (Wiler et al., 1995, Proc. Natl. Acad. Sci. 92:11485-11489). The repair of DSBs created during V(D)J recombination is essential for generating TCR chains (in T cells) and Ig proteins (in B cells). Thus, in scid animals both B and T cell development is arrested at an early stage resulting in immunodeficiency.
V(D)J recombination is responsible for forming the exons that encode the variable regions of TCR and immunoglobulin molecules (reviewed by Roth et al., 1995, Current Biology 5:496-499; Weaver, 1995, Trends in Genet. 11:388-392). Recombination is initiated by the introduction of DSBs at recombination signal sequences that are situated adjacent to the V, D, and J coding elements. Cleavage is performed by the RAG-1 and RAG-2 proteins and generates two types of broken ends: coding ends, which are covalently sealed in the form of hairpins (Roth et al., 1992, Cell 70:983-991; Zhu and Roth, 1995, Immunity 2:101-112) and signal ends, which are blunt (Roth et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:10788-10792; Schlissel et al., 1993, Genes Dev. 7:2520-2532). Joining of coding ends assembles the V, D, or J gene segments, forming a rearranged variable region exon (the junction is termed a coding joint) and joining of signal ends forms a reciprocal product termed a signal joint. Recent experiments both in cell-free systems (van Gent et al., 1995, Cell 81:925-934; McBlane et al., 1995, Cell 83:387-395) and in vivo (Ramsden and Gellert, 1995, Genes Dev. 9:2409-2420) have strongly suggested that blunt signal ends and hairpin coding ends are in fact normal intermediates in V(D)J recombination.
Although the characterization of DNA intermediates in vivo and the analysis of the cleavage reaction in vitro have provided important information about the roles of RAG-1 and RAG-2 in the cleavage reaction, the mechanisms responsible for joining signal and coding ends remain unknown. The mouse scid mutation blocks formation of coding joints and is characterized by accumulation of hairpin coding ends (Roth et al., 1992; Zhu and Roth, 1995).
The scid animals and cell lines are also hypersensitive to ionizing radiation due to a failure in repairing DSBs (Fulop and Phillips, 1990, Nature 347:479-482; Biedermann et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:1394-1397). This observation provided the first indication that there is overlap between general DSB repair pathways and V(D)J recombination, although subsequently several mutations that affect both processes have now been identified (see Roth et al., 1995 for review).
Recent work has shown that a single protein complex, the DNA-dependent protein kinase (DNA-PK) plays a critical role in both V(D)J recombination and DSB repair. The catalytic subunit of DNA-PK, DNA-PK.sub.cs, (an ATM homologue) was shown to be a strong candidate for the said defect in mice (Blunt et al., 1995, Cell 80:813-823; Kirchgessner et al., 1995, Science 267:1178-1182; Boubnov et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:890-894; Lees-Miller et al., 1995, Science 267:1183-1185) and horses (Wiler et al., 1995).
Additional mutations in cell lines that interfere with both V(D)J recombination and DSB repair were subsequently identified (Pergola et al., 1993, Mol. Cell. Biol. 13:3464-3471; Taccioli et al., 1993, Science 260:207-210). These cell lines comprised four complementation groups, termed XRCC4 through XRCC7. Three of these groups (XRCC5-7) were shown to represent each of the three components of DNA-PK. The observation that three of the four identified mutations that affect DSB repair and V(D)J recombination affect components of DNA-PK suggests that DNA-PK plays a critical role in these processes. DNA-PK is a trimeric molecule composed of Ku, a DNA binding heterodimer consisting of Ku70 and Ku86 subunits and DNA-PK.sub.cs. XRCC7 appears to encode DNA-PK.sub.cs (murine and foal scid). The sxi-1 cell line (XRCC6) can be complemented by a Ku70 cDNA (Weaver, 1995; Lee et al., 1995, Mutation Research 336:279-291). XRCC5 was rescued by Ku86 cDNA (Taccioli et al., 1994; Smider et al., 1994, Science 266:288-291; Boubnov et al., 1995) and genomic deletions were observed that removed part of the Ku86 coding sequence in XRCC5 cells (Errami et al., 1996, Mol. Cell. Biol. 16: 1519-1526). A deficiency in Ku86 resulted in unstable Ku70 that was rescued with expression of Ku86 cDNA (Errami et al., 1996). Therefore, a deficiency of Ku86 lead to a deficiency in Ku70 and a mutation in the sequences that code for either protein should effectively ablate the other. In addition, a Ku86 deficiency lead to no DNA-PK activity (Finnie et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:320-324). Therefore, a mutation in sequences that code for Ku86 should effectively ablate DNA-PK.sub.cs function; however, there may be Ku function that is independent of DNA-PK.sub.cs. The XRCC4 gene has recently been shown to encode a novel protein (Li et al., 1995, Cell 83:1079-1089), and xrcc4 mutant cell lines exhibit normal DNA-PK activity.
DNA-PK is activated by DNA lesions and is capable of phosphorylating a variety of substrates, including transcription factors and the p53 tumor suppressor protein. Upon Ku binding to DNA ends, the kinase activity becomes activated, leading to phosphorylation of a variety of protein substrates in vitro (Gottlieb and Jackson, 1994, Trends Biochem. Sci. 19:500-503). It has been suggested that DNA-PK may function to sense DNA damage (Roth et al., 1995). The suggestion that DNA-PK may be involved in cell cycle control fits with the recent observation that the catalytic subunit, DNA-PK.sub.cs, shares homology with PI 3-kinase family members involved in cell cycle control, DNA repair and DNA damage responses (reviewed by Zakian, 1995). This notion is supported by the observation that Ku deficient cells have a slightly longer cell division time, although no clear-cut cell cycle checkpoint deficiencies have been described (Jeggo, 1985, Mutation Research 145:171-176; Jeggo, 1990, Mutation Research 239:1-16; Lee et al., 1995, Mutation Research 336:279-291).
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.